Cell growth and differentiation are regulated by a large array of overlapping signalling pathways which coordinate both mRNA transcription and protein translation. While many individual details have been elucidated, it is clear that more information is necessary to fully understand the normal and aberrant regulation of these complex pathways. Many growth factors cause rapid and transient increases in intracellular calcium levels, however, the physiological consequences of this are not known. Calcium is widely recognized as an important messenger in eukaryotic systems. In many instances, its actions are mediated by binding to calmodulin, and strong evidence indicates, in turn, that the effects of calmodulin are often achieved through the regulation of protein phosphorylation. A potential target for this ubiquitous signalling pathway is elongation factor 2(EF-2), a key component in protein synthesis and hence cell growth. EF-2 is subject to two different types of post-translation modification. The protein is ADP-ribosylated by diphtheria toxin resulting in inhibition of protein synthesis and cell death. EF-2 is also phosphorylated by a specific calcium/calmodulin- dependent protein kinase, CaM kinase III, resulting in its complete inactivation. The exact physiological role of EF-2 phosphorylation is unknown; however, recent studies suggest a potential role in regulation of cell growth. EF-2 is rapidly phosphorylated in cells in response to a variety of growth factors and mitogens and this is associated with transient inhibition of protein synthesis. CaM kinase III levels are very high in rapidly growing cells but are down-regulated in non-dividing cells, and results using site-directed mutagenesis suggest that phosphorylation of EF-2 is necessary for cell viability. Finally, this signalling system is conserved in species as diverse as man and yeast. The overall aim of the proposed studies is to characterize the biochemical and physiological consequences of EF-2 phosphorylation. Specifically, the hypothesis will be tested in both mammalian and yeast cells, that EF-2 phosphorylation and inhibition of protein synthesis influences translation of specific proteins by affecting mRNA selection and/or mRNA stability. Results from these studies will lead to a greater understanding of the regulation by calcium of growth and differentiation in normal and cancerous cells.
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